#HIDDEN
from IPython.display import Image
from IPython.core.display import HTML
from ipywidgets import Text, Dropdown, Output, interact
from matplotlib import pyplot as plt
from pandas import DataFrame, merge, read_csv, Series, to_datetime
import cufflinks as cf
import qgrid
cf.go_offline()
# cf.set_config_file(offline=False, world_readable=True)
cf.set_config_file(offline=True, world_readable=True)
###  nbi:hide_in

MetaG Sequencing and Alignment Summaries

Full mapping script

Adapter Trimming and QC (Trimmomatic) Report

What is the abundance and quality of the reads in each sample?

Step 1. Split JGI files in PE files

split-paired-reads.py --gzip -1 \$sample.fastq.pe1.gz -2 \$sample.fastq.pe2.gz \$jgi_combined_fastq

Step 2. Use trimmomatic to QC reads
trimmomatic PE -phred33 \$sample.fastq.pe1.gz \$sample.fastq.pe2.gz \$sample.fastq.pe1.gz \$sample.fastq.se1.gz \$sample.fastq.pe2.gz \$sample.fastq.se2.gz \
   ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10:8:TRUE \
   LEADING:3 \
   TRAILING:3 \
   SLIDINGWINDOW:4:15 \
   MINLEN:36

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Or skip to:
#HIDDEN
# <!-- # #HIDDEN
# # tImages = {"MetaG":Image("images/TrimReport_MetaG.png"),"MetaT":Image("images/MetaT_trimmomatic_plot.png")}
# # @interact
# # def trimReport(Source=["MetaG","MetaT"]):return tImages[Source] 
# <img src="images/TrimReport_MetaG.png"></img>
# -->
HTML("html/MetaG_TrimStats.html")
MultiQC Report

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        MultiQC: Summarize analysis results for multiple tools and samples in a single report
        Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
        Bioinformatics (2016)
        doi: 10.1093/bioinformatics/btw354
        PMID: 27312411

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        About MultiQC

        This report was generated using MultiQC, version 1.7

        You can see a YouTube video describing how to use MultiQC reports here: https://youtu.be/qPbIlO_KWN0

        For more information about MultiQC, including other videos and extensive documentation, please visit http://multiqc.info

        You can report bugs, suggest improvements and find the source code for MultiQC on GitHub: https://github.com/ewels/MultiQC

        MultiQC is published in Bioinformatics:

        MultiQC: Summarize analysis results for multiple tools and samples in a single report
        Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
        Bioinformatics (2016)
        doi: 10.1093/bioinformatics/btw354
        PMID: 27312411

        Report generated on 2019-10-27, 12:13 based on data in: /mnt/research/ShadeLab/GLBRC/mapping/metaG/fullAssembly/trimStats


        Trimmomatic is a flexible read trimming tool for Illumina NGS data.

        Host Plant Alignment Report

        When the reads are aligned to plant reference genomes, how many of the reads are plant-aligned reads?

        Step 1. Align reads to respective host reference assembly

        if [[ \$sample == *"G5"* ]]; then
               SAMFILE=\$sample.SWGRASS.sam
               BAMFILE=\$sample.SWGRASS.bam
               bowtie2 --threads \$THREADS -x \$SWITCHGRASS
                     -1 \$sample.fastq.pe1.gz -2 \$sample.fastq.pe2.gz
                     -U \$sample.fastq.se12.gz -S \$sample.SWGRASS.sam 2>\$sample.stat
        else
               SAMFILE=\$sample.MISCANTS.sam
               BAMFILE=\$sample.MISCANTS.bam
               bowtie2 --threads \$THREADS -x \$MISCANTHUS
                     -1 \$sample.fastq.pe1.gz -2 \$sample.fastq.pe2.gz
                     -U \$sample.fastq.se12.gz -S \$sample.MISCANTS.sam 2>\$sample.stat
        fi

        #HIDDEN
        HTML(filename="html/BothPerc.html")
        

        Sequencing and alignment Summaries

        Fungal Alignment Report

        What reads are fungal reads?


        Step 1. Extract reads that don't align the plant assembly

        # A. Convert sam > bam
        samtools view -bS \$SAMFILE > \$BAMFILE

        # B. Extract all unmapped reads (-f 4) that don't have a mate mapped (-F 256 i.e. both unmapped)
        samtools view -b -f 4 -F 256 \$BAMFILE > \$sample.unmapped.bam

        # C. Sort the reads
        samtools sort -n \$sample.unmapped.bam -o \$sample.unmapped_sorted.bam

        # D. Split the unmapped reads in R1 & R2 (back into paired end)
        bedtools bamtofastq -i \$sample.unmapped_sorted.bam -fq \$sample.R1.fastq -fq2 \$sample.R2.fastq

        Step 2. Align the reads to the combined fungal assemblies
        bowtie2 -x \$FUNGAL -1 \$sample.R1.fastq -2 \$sample.R2.fastq -S \$sample.fungal.sam 2>\$sample.fungal.stat

        #HIDDEN
        HTML(filename='html/FungalAlign.html')
        

        Sequencing and alignment Conclusions

        Sequencing Observations

        What patterns are there over the season?
        1. The percentage of plant reads sequenced is high thoughout the season and tapers off towards the end of the season. This may be do to senescence of plant cells.
        2. Alignment to the 6 selected fungal assemblies is low, but picks up during the warmer months. Overall, reads aligning to the combined fungal assembly were <10% of the non-plant reads.

        Annotation Summary

        The annotations were performed using KEGG's prokaryotic peptides and eukaryotic peptides

        Based on the high-overlap, we decided to use the mags instead of the assembly for alignment

        MetaG Analysis

        MAG Stats

        MAGs generated with reads that did not align to the fungal/host assemblies using metabat. Stats were then generated with checkm and the command below, results for that stats are below that. checkm lineage_wf -t 20 -x fa Final.contigs.fa.metabat-bins20 metaBinsStats
        #HIDDEN
        HTML(filename="html/MAG_Stats.html")
        

        Table of the bins with ≥ 50% completeness

        Bin Id Marker lineage Unnamed: 2 # Genomes # Markers # Marker sets 0 1 2 3 4 5+ Completeness Contamination Strain heterogeneity
        0 bin.7 o__Burkholderiales (UID4105) 54 553 264 3 534 16 0 0 0 99.66 2.65 43.75
        1 bin.39 f__Enterobacteriaceae (UID5054) 223 874 303 38 829 7 0 0 0 99.05 0.70 71.43
        2 bin.67 o__Pseudomonadales (UID4488) 185 812 308 35 751 26 0 0 0 97.79 3.53 46.15
        3 bin.157 o__Actinomycetales (UID1593) 69 400 198 21 364 14 1 0 0 96.84 4.73 29.41
        4 bin.125 o__Actinomycetales (UID1663) 488 310 185 18 276 16 0 0 0 93.96 4.23 68.75
        5 bin.164 o__Burkholderiales (UID4000) 193 427 214 44 379 4 0 0 0 92.27 1.64 50.00
        6 bin.117 o__Rhizobiales (UID3654) 92 481 319 66 401 14 0 0 0 90.51 3.00 78.57
        7 bin.202 o__Cytophagales (UID2936) 47 454 336 82 362 10 0 0 0 89.90 2.68 80.00
        8 bin.181 o__Rhizobiales (UID3654) 92 481 319 73 381 27 0 0 0 89.84 5.71 33.33
        9 bin.188 o__Burkholderiales (UID4000) 193 427 214 77 296 51 3 0 0 87.56 15.48 60.00
        10 bin.198 o__Cytophagales (UID2936) 47 454 336 54 379 21 0 0 0 87.28 3.43 61.90
        11 bin.121 o__Burkholderiales (UID4000) 193 427 214 68 339 20 0 0 0 87.25 5.02 30.00
        12 bin.95 o__Burkholderiales (UID4002) 107 574 251 67 493 14 0 0 0 87.18 2.41 78.57
        13 bin.214 o__Flavobacteriales (UID2815) 123 324 204 67 252 5 0 0 0 87.11 1.49 100.00
        14 bin.195 k__Bacteria (UID203) 5449 104 58 25 54 16 8 1 0 82.99 35.50 78.26
        15 bin.105 o__Actinomycetales (UID1593) 69 400 198 104 282 14 0 0 0 75.84 4.46 28.57
        16 bin.88 k__Bacteria (UID203) 5449 104 58 28 39 22 10 5 0 73.82 38.55 34.15
        17 bin.142 c__Deltaproteobacteria (UID3216) 83 247 155 49 194 4 0 0 0 73.12 2.58 25.00
        18 bin.148 o__Sphingomonadales (UID3310) 26 569 293 154 398 17 0 0 0 72.70 2.26 17.65
        19 bin.112 o__Flavobacteriales (UID2815) 123 324 204 100 214 10 0 0 0 71.14 2.71 80.00
        20 bin.91 o__Sphingomonadales (UID3310) 26 569 293 191 261 99 18 0 0 69.76 25.37 22.22
        21 bin.206 o__Rickettsiales (UID3809) 83 324 211 86 226 12 0 0 0 69.27 3.82 58.33
        22 bin.93 o__Actinomycetales (UID1802) 274 385 212 105 254 25 1 0 0 68.43 8.09 53.57
        23 bin.114 o__Actinomycetales (UID1663) 488 310 185 92 215 3 0 0 0 67.03 0.69 33.33
        24 bin.104 k__Bacteria (UID203) 5449 103 57 52 38 12 1 0 0 67.01 12.41 46.67
        25 bin.134 o__Rhizobiales (UID3654) 92 481 319 183 288 10 0 0 0 66.91 2.00 80.00
        26 bin.163 o__Rickettsiales (UID3809) 83 324 211 101 216 7 0 0 0 66.80 2.53 14.29
        27 bin.146 o__Actinomycetales (UID1697) 387 330 193 131 190 9 0 0 0 64.35 2.27 0.00
        28 bin.205 o__Actinomycetales (UID1697) 387 330 193 125 198 7 0 0 0 63.48 3.11 42.86
        29 bin.200 k__Bacteria (UID203) 5449 104 58 59 43 2 0 0 0 62.47 2.59 100.00
        30 bin.57 o__Cytophagales (UID2936) 47 454 336 192 256 6 0 0 0 60.73 1.79 50.00
        31 bin.10 k__Bacteria (UID203) 5449 104 58 63 41 0 0 0 0 60.69 0.00 0.00
        32 bin.208 c__Alphaproteobacteria (UID3305) 564 337 221 134 193 10 0 0 0 60.38 2.96 90.00
        33 bin.2 o__Actinomycetales (UID1815) 120 574 266 221 331 22 0 0 0 59.37 3.03 36.36
        34 bin.175 o__Burkholderiales (UID4000) 193 427 214 171 246 8 2 0 0 58.90 2.35 50.00
        35 bin.116 k__Bacteria (UID203) 5449 104 58 61 31 11 0 1 0 58.79 18.97 100.00
        36 bin.66 o__Sphingomonadales (UID3310) 26 569 293 263 287 17 2 0 0 56.39 5.49 52.17
        37 bin.139 o__Actinomycetales (UID1593) 69 400 198 165 203 31 0 1 0 56.31 7.46 64.86
        38 bin.156 k__Bacteria (UID203) 5449 104 58 60 39 5 0 0 0 56.04 6.19 100.00
        39 bin.55 k__Bacteria (UID203) 5449 103 57 65 28 9 1 0 0 56.01 13.16 66.67
        40 bin.84 o__Burkholderiales (UID4000) 193 427 214 212 206 9 0 0 0 55.53 2.95 11.11
        41 bin.171 k__Bacteria (UID203) 5449 104 58 35 31 24 14 0 0 55.22 22.74 3.03
        42 bin.132 f__Rhizobiaceae (UID3564) 78 840 354 352 470 18 0 0 0 52.64 1.16 61.11
        43 bin.54 k__Bacteria (UID203) 5449 103 57 69 34 0 0 0 0 52.63 0.00 0.00
        44 bin.199 k__Bacteria (UID203) 5449 104 58 65 37 2 0 0 0 52.47 3.45 50.00
        45 bin.115 k__Bacteria (UID203) 5449 104 58 43 38 12 11 0 0 52.30 19.17 66.67
        46 bin.120 o__Sphingomonadales (UID3310) 26 569 293 274 271 23 1 0 0 51.10 5.80 38.46
        47 bin.33 k__Bacteria (UID203) 5449 104 58 68 36 0 0 0 0 50.17 0.00 0.00
        48 bin.65 k__Bacteria (UID203) 5449 104 58 64 39 1 0 0 0 49.32 1.72 100.00

        MetaT Analysis

        Overarching analysis - changes in metagenome content over time

        All bins with at least 50% completeness were used in a combined assembly. The analysis below comes from the counts for each sample. These counts were normalized by the number of reads that did not align to the fungal or host assemblies.

        COG accumulation over time

        pcoa

        Is time a factor that affects the functional richness of phylosphere communities (Alpha Diversity)?

        Switchgrass Pearson's product-moment correlation
        Data: Time and Richness
        t = 10.223 df = 62 p-value = 6.316e-15
        sample estimates: cor 0.7922484
        Switchgrass Pearson's product-moment correlation
        Data: Time and Shannon
        t = 7.1534 df = 62 p-value = 1.166e-09
        sample estimates: cor 0.6724267
        Switchgrass Pearson's product-moment correlation
        Data: Time and Pielou
        t = 5.6951 df = 62 p-value = 3.622e-07
        sample estimates: cor 0.586056
        Miscanthus Pearson's product-moment correlation
        Data: Time and Richness
        t = 8.5659 df = 70 p-value = 1.658e-12
        sample estimates: cor 0.7153784
        Miscanthus Pearson's product-moment correlation
        Data: Time and Shannon
        t = 6.1267 df = 70 p-value = 4.675e-08
        sample estimates: cor 0.5908107
        Miscanthus Pearson's product-moment correlation
        Data: Time and Pielou
        t = 7.1602 df = 70 p-value = 6.369e-10
        sample estimates: cor 0.6502079

        Do we see changes over time in functional richness?

        Call: adonis(formula = dist.otu ~ map_16S\$time_numeric)

        Permutation: free Number of permutations: 999

        Terms added sequentially (first to last)

        Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
        map_16S$time_numeric 1 1.9356 1.93562 47.814 0.26298 0.001 ***
        Residuals 134 5.4246 0.04048 0.73702
        Total 135 7.3603 1.000000

        images/Level1_Mags.png

        images/Level2_Mags.png

        Is there a difference between treatment (Fertilized vs. Unfertilized)?

        Call: adonis(formula = dist.otu ~ map_16S\$treatment)

        Permutation: free Number of permutations: 999

        Terms added sequentially (first to last)

        Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
        map_16S$treatment 1 0.0289 0.028930 0.52878 0.00393 0.755
        Residuals 134 7.3313 0.054711 0.99607
        Total 135 7.3603 1.000000

        Is there a difference between host plant?

        Call: adonis(formula = dist.otu ~ map_16S\$plant)

        Permutation: free Number of permutations: 999

        Terms added sequentially (first to last)

        Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
        map_16S$plant 1 0.1617 0.161704 3.0101 0.02197 0.027 *
        Residuals 134 7.1986 0.053721 0.97803
        Total 135 7.3603 1.000000

        Is there similarity between the WGS communities and the 16S data (mantel/procrustes)?

        Mantel statistic based on Pearson's product-moment correlation Call: mantel(xdis = genes.dist, ydis = otu.dist) Mantel statistic r: 0.6526 Significance: 0.001 Upper quantiles of permutations (null model): 90% 95% 97.5% 99% 0.0833 0.1194 0.1458 0.1849 Permutation: free Number of permutations: 999 Call: protest(X = genes.pcoa, Y = otu.pcoa) Procrustes Sum of Squares (m12 squared): 0.5402 Correlation in a symmetric Procrustes rotation: 0.6781 Significance: 0.001 Permutation: free Number of permutations: 999

        How variable are metagenomes across replicate time points?

         
        

        Metatranscriptomic Sequencing

        MetaT Sequencing and Alignment Summaries

        Full mapping script

        What is the abundance and quality of the reads in each sample?

        #HIDDEN
        HTML("html/MetaT_TrimStats.html")
        
        MultiQC Report

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              MultiQC: Summarize analysis results for multiple tools and samples in a single report
              Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
              Bioinformatics (2016)
              doi: 10.1093/bioinformatics/btw354
              PMID: 27312411

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              About MultiQC

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              For more information about MultiQC, including other videos and extensive documentation, please visit http://multiqc.info

              You can report bugs, suggest improvements and find the source code for MultiQC on GitHub: https://github.com/ewels/MultiQC

              MultiQC is published in Bioinformatics:

              MultiQC: Summarize analysis results for multiple tools and samples in a single report
              Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
              Bioinformatics (2016)
              doi: 10.1093/bioinformatics/btw354
              PMID: 27312411

              Report generated on 2020-02-25, 12:44 based on data in: /mnt/research/ShadeLab/GLBRC/mapping/metaT/fullAssembly/trimStats


              loading..

              How do the reads align to the Switchgrass assembly?

              Season Trend

              #HIDDEN
              HTML("html/MetaT_HostAlignment.html")
              
              MultiQC Report

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                    MultiQC: Summarize analysis results for multiple tools and samples in a single report
                    Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
                    Bioinformatics (2016)
                    doi: 10.1093/bioinformatics/btw354
                    PMID: 27312411

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                    About MultiQC

                    This report was generated using MultiQC, version 1.7

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                    For more information about MultiQC, including other videos and extensive documentation, please visit http://multiqc.info

                    You can report bugs, suggest improvements and find the source code for MultiQC on GitHub: https://github.com/ewels/MultiQC

                    MultiQC is published in Bioinformatics:

                    MultiQC: Summarize analysis results for multiple tools and samples in a single report
                    Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
                    Bioinformatics (2016)
                    doi: 10.1093/bioinformatics/btw354
                    PMID: 27312411

                    Report regenerated on 2020-02-18, 21:40 based on data in: /mnt/research/ShadeLab/GLBRC/mapping/metaT/hostRemovalFlagstats


                    There are 6 possible types of alignment: * PE mapped uniquely: Pair has only one occurence in the reference genome. * PE mapped discordantly uniquely: Pair has only one occurence but not in proper pair. * PE one mate mapped uniquely: One read of a pair has one occurence. * PE multimapped: Pair has multiple occurence. * PE one mate multimapped: One read of a pair has multiple occurence. * PE neither mate aligned: Pair has no occurence.

                    loading..

                    How do the reads align to the MAG assembly?

                    HTML("html/MetaT_MAG_Align.html")
                    
                    MultiQC Report

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                          MultiQC: Summarize analysis results for multiple tools and samples in a single report
                          Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
                          Bioinformatics (2016)
                          doi: 10.1093/bioinformatics/btw354
                          PMID: 27312411

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                          MultiQC is published in Bioinformatics:

                          MultiQC: Summarize analysis results for multiple tools and samples in a single report
                          Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
                          Bioinformatics (2016)
                          doi: 10.1093/bioinformatics/btw354
                          PMID: 27312411

                          Report generated on 2020-02-18, 15:40 based on data in: /mnt/research/ShadeLab/GLBRC/mapping/metaT/magFlagstats


                          This plot shows the number of reads aligning to the reference in different ways.
                          Please note that single mate alignment counts are halved to tally with pair counts properly.

                          There are 6 possible types of alignment: * PE mapped uniquely: Pair has only one occurence in the reference genome. * PE mapped discordantly uniquely: Pair has only one occurence but not in proper pair. * PE one mate mapped uniquely: One read of a pair has one occurence. * PE multimapped: Pair has multiple occurence. * PE one mate multimapped: One read of a pair has multiple occurence. * PE neither mate aligned: Pair has no occurence.

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                                Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
                                Bioinformatics (2016)
                                doi: 10.1093/bioinformatics/btw354
                                PMID: 27312411

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                                For more information about MultiQC, including other videos and extensive documentation, please visit http://multiqc.info

                                You can report bugs, suggest improvements and find the source code for MultiQC on GitHub: https://github.com/ewels/MultiQC

                                MultiQC is published in Bioinformatics:

                                MultiQC: Summarize analysis results for multiple tools and samples in a single report
                                Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
                                Bioinformatics (2016)
                                doi: 10.1093/bioinformatics/btw354
                                PMID: 27312411

                                Report generated on 2020-02-25, 12:44 based on data in: /mnt/research/ShadeLab/GLBRC/mapping/metaT/fullAssembly/trimStats

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                                Outline

                                Research Questions

                                 
                                

                                Next Steps

                                1. Fix the metadata to ensure that each sample is accounted for and we have the seq files (Nejc)      ✓ Completed Wednesday, Feb 19,2020. Delivered by Kiera
                                2. Modify the R analysis script to read results from the MetaT data (Nejc & Shane)

                                  In order for this to progress, 1 had to be complete. The next step is figure out why the metadata for the MetaT is not meshing with the R script.

                                3. Explore the Kallisto output to determine how to extract annotations (Shane)

                                  Kallisto has an arguement to export a bam file when it runs, but I do not know how these bam files work or if they are the same thing as a bam from bowtie2. I am working with kallisto to get a bam file now and then I will see if that file works to extract annotations from the contigs